U.S. patent number 10,527,544 [Application Number 15/735,661] was granted by the patent office on 2020-01-07 for atr-ftir for non-invasive detection of colitis.
This patent grant is currently assigned to GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC., THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS. The grantee listed for this patent is THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS, GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC., THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF VETERANS AFFAIRS. Invention is credited to Merlin Didier, A. G. Unil Perera, Jitto Titus, Emilie Viennois.
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United States Patent |
10,527,544 |
Titus , et al. |
January 7, 2020 |
ATR-FTIR for non-invasive detection of colitis
Abstract
Disclosed are methods, systems, and apparatuses for non-invasive
detection of colitis in a subject. The methods involve depositing a
bodily fluid sample from the subject on an internal reflection
element (IRE). A beam of infrared (IR) radiation can then be
directed through the IRE under conditions such that the IR
radiation interacts with the bodily fluid sample. An absorption
spectrum can then be recorded over a range of preselected
frequencies to detect peaks that are affected by colitis. In
preferred embodiments, the methods and systems involve Fourier
Transform Infrared Spectroscopy (FTIR).
Inventors: |
Titus; Jitto (Acworth, GA),
Viennois; Emilie (Atlanta, GA), Perera; A. G. Unil
(Mableton, GA), Didier; Merlin (Decatur, GA) |
Applicant: |
Name |
City |
State |
Country |
Type |
GEORGIA STATE UNIVERSITY RESEARCH FOUNDATION, INC.
THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF
VETERANS AFFAIRS |
Atlanta
Washington |
GA
DC |
US
US |
|
|
Assignee: |
GEORGIA STATE UNIVERSITY RESEARCH
FOUNDATION, INC. (Atlanta, GA)
THE UNITED STATES GOVERNMENT AS REPRESENTED BY THE DEPARTMENT OF
VETERANS AFFAIRS (Washington, DC)
|
Family
ID: |
57504354 |
Appl.
No.: |
15/735,661 |
Filed: |
June 13, 2016 |
PCT
Filed: |
June 13, 2016 |
PCT No.: |
PCT/US2016/037172 |
371(c)(1),(2),(4) Date: |
December 12, 2017 |
PCT
Pub. No.: |
WO2016/201408 |
PCT
Pub. Date: |
December 15, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20180364163 A1 |
Dec 20, 2018 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62175050 |
Jun 12, 2015 |
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62321542 |
Apr 12, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
21/552 (20130101); A61B 5/4255 (20130101); G01N
21/3581 (20130101); G01N 2800/102 (20130101); G01N
2021/3595 (20130101); G01N 2800/067 (20130101) |
Current International
Class: |
G01N
21/552 (20140101); G01N 21/3581 (20140101); A61B
5/00 (20060101); G01N 21/35 (20140101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-9517092 |
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Jun 1995 |
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WO |
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2009/121054 |
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Oct 2009 |
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WO |
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2014/076480 |
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May 2014 |
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WO |
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2015/085056 |
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Jun 2015 |
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WO |
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Other References
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biomarkers in inflammatory bowel disease" Journal of proteomics
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Microbiota in Mice Lacking Toll-Like Receptor 5" Science, 2010.
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Microorganisms by Fourier Transform Infrared Microspectroscopy"
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Mucous Membrane of the Lip: Application of a Chalcogenide Optical
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cited by applicant.
|
Primary Examiner: LaPage; Michael P
Attorney, Agent or Firm: Meunier Carlin & Curfman
LLC
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government Support under Grant Nos.
DK071594 and DK064711 awarded by the National Institutes of Health.
The Government has certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a national stage application filed under 35
U.S.C. .sctn. 371 of PCT/US2016/037172, which claims benefit of
U.S. Provisional Application No. 62/175,050, filed Jun. 12, 2015,
and application Ser. No. 62/321,542, filed Apr. 12, 2016, which are
hereby incorporated herein by reference in their entirety.
Claims
What is claimed is:
1. A method for detecting inflammation in a subject, comprising:
(a) depositing a bodily fluid sample from the subject on an
internal reflection element (IRE); (b) directing a beam of infrared
(IR) radiation through the IRE under conditions such that the IR
radiation interacts with the bodily fluid sample; (c) recording an
absorption spectrum over a range of preselected frequencies; (d)
comparing the absorption spectrum to a control spectrum to identify
spectral events associated with inflammation; and (e) treating the
subject for colitis if an increase in the ratio of 1650:1635
cm.sup.-1 peaks compared to a normal control is detected; or
treating the subject for arthritis if peaks are detected at 1033
cm.sup.-1 but not 1076 cm.sup.-1; or treating the subject for
arthritis if peaks are detected at 1292 cm.sup.-1.
2. The method of claim 1, wherein the IRE is an attenuated total
reflectance (ATR) crystal comprising an optical material with a
higher refractive index than the sample.
3. The method of claim 2, wherein the IRE comprises a germanium
crystal or a zinc selenide crystal.
4. The method of claim 1, wherein the IR radiation that interacts
with the bodily fluid sample is an evanescent wave with an average
penetration depth of about 2 .mu.m.
5. The method of claim 1, further comprising Fourier transformation
of the absorbance spectrum.
6. The method of claim 1, wherein the range of preselected
frequencies is between 50 cm.sup.-1 and 3700 cm.sup.-1.
7. The method of claim 1, wherein the bodily fluid sample comprises
a blood, serum, or plasma sample.
Description
BACKGROUND
Inflammatory disorders of the gastrointestinal tract are caused due
to environmental or genetic factors. Some of the Inflammatory bowel
diseases (IBD) such as ulcerative colitis (Kornbluth A, et al. J
Clin Gastroenterol. 1995 20(4):280-4) and Crohn's disease
(Friedman, S, et al. Gastroenterology. 2001 120(4):820-6) are
debilitating and can lead to life threatening complications such as
colorectal cancer (Argov, S, et al. Biopolymers. 2004
75(5):384-92). Assessment of intestinal inflammation in IBD remains
a difficult challenge (Schreyer, A, et al. Gut. 2005 54(2):250-6).
Currently, the clinical diagnosis of IBD is achieved through
colonoscopy, which is used to assess the endoscopic appearance of
the colon. However, this technique is not ideal for monitoring
disease activity regularly or as an annual checkup and is
expensive, invasive requiring sedation with probable complications.
Thus, there is a need for new, low risk, simple, inexpensive and
objective tools for IBD diagnostics especially for annual
checkups.
SUMMARY
Disclosed are methods, systems, and devices for non-invasive
detection of inflammatory disease, e.g., colitis, in a subject. The
disclosed method can involve depositing a sample from the subject
on an internal reflection element (IRE). In some embodiments the
sample is allowed to dry. A beam of infrared (IR) radiation can
then be directed through the IRE under conditions such that the IR
radiation interacts with the bodily fluid sample. In some
embodiments, the IR radiation is an evanescent wave with an average
penetration depth of about 2 .mu.m. An absorption spectrum can then
be recorded over a range of preselected frequencies. This
absorption spectrum can then be compared to a control spectrum to
identify spectral events associated with colitis.
In some embodiments, the IRE is an attenuated total reflectance
(ATR) crystal comprising an optical material with a higher
refractive index than the sample comprising the plurality of cells.
For example, the IRE can be a germanium, zinc selenide, silicon,
diamond, or KRS-5 crystal.
In preferred embodiments, the methods and systems involve Fourier
Transform Infrared Spectroscopy (FTIR). Therefore, the disclosed
methods and systems can further comprise Fourier transformation of
the absorbance spectrum. In some embodiments, the ATR crystal is
used with a diffractive monochromator instead of an FTIR.
The range of preselected frequencies for recording absorbance can
be between 50 cm.sup.-1 and 3700 cm.sup.-1. In some embodiments,
peaks at approximately 1033 cm.sup.-1, 1076 cm.sup.-1, 1292
cm.sup.-1, 1704 cm.sup.-1, or a combination thereof, are an
indication of colitis in the subject. Subjects with colitis have
higher absorbance at spectral markers 1704 cm.sup.-1, 1033
cm.sup.-1 and 1076 cm.sup.-1. An increase in absorbance is seen in
arthritic sera samples at 1704 cm.sup.-1 and 1033 cm.sup.-1
(similar to colitic samples), but not at 1076 cm.sup.-1. Arthritis
can be specifically detected by monitoring absorbance at 1292
cm.sup.-1.
In some embodiments, the sample is bodily fluid sample from a
subject suspected of having an inflammatory disease or disorder,
e.g., colitis or other inflammatory bowel disorder. The disclosed
method can further involve examining the subject by colonoscopy or
treating the subject if colitis is indicated.
The details of one or more embodiments of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1A shows Lcn-2 quantified in the feces of mice showing a clear
increase of Lcn-2 in colitic IL10-/- vs. non-colitic IL10-/- mice.
FIG. 1B shows colonic myeloperoxidase (MPO) activity quantified in
the distal colon of DSS induced-colitis compared to water control
mice agreeing well with the spectroscopy data. FIG. 1C shows
respective H&E-stained colons of WT water control, DSS-induced
colitis and colitic IL10-/- mice indicate sites of lymphocytes
infiltrations (arrow heads) and erosion of the crypt figures
(arrows). Scale bar: 100 .mu.m.
FIG. 2 shows averaged ATR-FTIR spectra of sera drawn from mice
before (n=12) and after (n=12) developing colitis induced by 3%
DSS. The differentiating markers 1033 and 1076 cm.sup.-1 are
identified as glucose and mannose with p-values of 4.43 E-8 and
7.59 E-8 respectively. The inset shows the individual serum samples
from 1140-1000 cm.sup.-1 for clarity. Individual colitic and
non-colitic spectra show a clear separation between the groups.
With further data points it should be possible to find an
absorbance range for the two groups. All spectra are normalized to
the Amide I peak (1642 cm.sup.-1). The averages for the glucose
peak are 0.3175.+-.0.0024 (non-colitic) and 0.3788.+-.0.0041
(colitic) and the averages for the mannose peak are
0.3847.+-.0.0022 (non-colitic) and 0.438.+-.0.0035 (colitic).
FIG. 3 shows averaged ATR-FTIR spectra of sera drawn from IL10-/-
mice before (n=4) and after (n=4) spontaneously developing colitis.
The same markers 1033 and 1076 cm.sup.-1 identified in the DSS
model are effective in differentiating colitic from non-colitic
spectra of the IL10-/- model. The inset shows the individual serum
samples from 1140-1000 cm.sup.-1 for clarity, again showing a clear
separation between the two groups. All spectra are normalized to
the Amide I peak (1642 cm.sup.-1). The averages for the glucose
peak are 0.3491.+-.0.0057 (non-colitic) and 0.412.+-.0.009
(colitic) and the averages for the mannose peak are
0.4071.+-.0.0034 (non-colitic) and 0.4553.+-.0.0081 (colitic).
FIG. 4 shows ATR-FTIR spectra of sera drawn from mice before (n=4)
and after (n=4) developing metabolic syndrome. In these 8 spectra,
the two spectral markers at 1033 cm.sup.-1 and 1076 cm.sup.-1 do
not show any difference in the metabolic syndrome samples with
respect to ATR-FTIR technique. The inset shows the spectra
(1140-1000 cm.sup.-1) of sera drawn from collagen antibody-induced
arthritic (n=4) and normal (n=4) mice (total of 8). 1033 cm.sup.-1
marker is common to colitis and arthritis, but 1076 cm.sup.-1
marker is unique to colitis. All spectra are normalized to the
Amide I peak (1642 cm.sup.-1).
FIGS. 5A and 5B show plots of the absorbances for the glucose peak
(FIG. 5A) at .about.1033 cm.sup.-1 and the mannose peak (FIG. 5B)
at .about.1076 cm.sup.-1 for Colitic (DSS), Colitic (IL10-/-),
Arthritic (CAIA) and Metabolic syndrome samples. FIGS. 5C and 5D
show the average values of absorbances for the normal and diseased
samples with the error bars. The error bars associated with the
normal samples are much smaller than the diseased samples as
expected. The metabolic syndrome samples do not show a separation
from the normal at either of the two peaks. However especially for
colitis samples, there is a clear separation from the normal
samples. The absorbance data associated with the peak at
.about.1033 cm.sup.-1 for arthritis also show a separation but not
at .about.1076 cm.sup.-1. Hence this analysis shows that the
absorbance data related to the mannose peak at .about.1076
cm.sup.-1 is unique to colitis.
FIG. 6 shows second derivative of the absorbances of colitic
(IL10-/- and DSS), metabolic syndrome and arthritic samples clearly
indicating the 1292 cm.sup.-1 peak identified as thymine which is
unique to arthritis.
FIG. 7 shows dendrogram plots of the cluster analyses of colitis
DSS sample spectra (12 colitic and 12 control) in the range of 1140
to 1000 cm.sup.-1 to include glucose (1033 cm.sup.-1) and mannose
(1076 cm.sup.-1) peaks. The spectra are correctly classified into
the colitic and control groups based on their conformity to each
other. Large heterogeneity is seen between colitis and control
samples (2.5) indicating that the two groups are distinctly
different. Similar heterogeneity (1.3) is seen in the IL10-/-
study.
FIG. 8 shows spectra of mice sera before and after developing
colitis employing the DSS (n=24) and IL10-/- (n=6) models.
Extra-intestinal inflammatory controls namely arthritis and
metabolic syndrome were also studied. The signatures (1033 and 1076
cm.sup.-1) differentiating colitic from non-colitic are identified
as glucose and mannose. DSS study had p-values of 4.43 E-8 and 7.59
E-8 at glucose and mannose peaks respectively. Inset (table) shows
that the glucose signature is common to colitis and arthritis, but
mannose signature is unique to colitis. Arthritis has a unique
signature at 1292 cm.sup.-1 which is identified as thymine (see
FIG. 4). All spectra are normalized to the Amide I peak (1642
cm.sup.-1).
FIG. 9 shows second derivative of the absorbance at the amide I
region indicates a significant difference between normal and
colitic samples while considering the alpha helix to beta sheet
ratios. As seen in the inset, there is no such difference for
arthritic model which serves as an extra-inflammatory control for
colitis. Thus, the alpha helix:beta sheet (1650:1635 cm.sup.-1)
ratio serves as a screening signature for colitis.
FIG. 10 shows the spectrum of normal mouse serum with the major
peaks assigned. Inset (a) shows the schematic working of ATR
technique. Spectra can be normalized to Amide I peak at 1642
cm.sup.-1. The Lorentzian oscillators for amide I and
polysaccharides (inset b) and the fittings for the experimental
absorbance curve are also shown. The oscillators used are 1: phenyl
ring stretch, 2: .alpha.-helix, 3: .beta.-sheet, 4 & 5:
carbohydrates, 6: mannose, 7: glucose.
DETAILED DESCRIPTION
The disclosed methods involve the use of an infrared spectrum
measuring apparatus. In some embodiments, the apparatus comprises:
an internal reflecting element (IRE) comprising a reflection face
located on the IRE at a region of intended contact between the IRE
and a sample; an infrared radiation source for supplying an
evanescent wave of infrared radiation and directing the same from
the outside of the IRE to the inside thereof so as to cause the
infrared radiation to be incident on the reflection face; and a
detector for detecting the once-reflected infrared radiation.
Representative, but non-limiting examples of instruments that can
provide the infrared radiation source include Fourier Transform
Infrared Spectroscopy (FTIR) spectrometers.
The term "internal reflection element" or IRE refers to a crystal,
prism, or other structure that will admit incoming radiation and
reflect the radiation at least once from a surface on the interior
of the element, preferably following interaction of the radiation
with a sample in contact with the reflecting surface. Following
such a reflectance, the radiation can be re-reflected or emitted
from the element. Preferably the IRE comprises a germanium crystal,
a zinc selenide crystal, or other material with higher index of
refraction than the refractive index of the sample being read that
are capable of transmitting IR or visible light.
The term "multi-pass ATR" refers to an attenuated total reflectance
technique in which radiation that is incident on an internal
reflectance element having two or more reflection faces within the
IRE experiences two or more interactions with a reflection face
before exiting the IRE. At these interfaces, the light is totally
reflected back into the IRE material. Such interactions are
typically referred to as "bounces" or "passes". Application of
multi-pass ATR generates a multi-pass ATR spectrum. Typically, the
IRE is in contact with a sample, the incident radiation is IR
radiation and the exiting radiation subsequently interacts with a
detector.
The term "single-pass ATR" refers to an attenuated total
reflectance technique in which radiation incident on an internal
reflectance element (IRE) having one or more reflection faces
within the IRE experiences only one interaction with a reflection
face before exiting the IRE. At this interface, the light is
totally reflected back into the IRE material. Application of
single-pass ATR generates a single-pass ATR spectrum.
The term "reflecting surface" refers to a surface capable of
reflecting incident radiation. On the IR surface where the sample
is deposited, the incident light is at an angle greater than the
critical angle and hence experiences total internal reflection.
There is no transmission of light at this interface, but rather an
evanescent wave that escapes out of the surface of the IRE but is
coupled back into the IRE material. Indeed, the technique of
attenuated total internal reflection (ATR) is based on the
principle that an evanescent wave interacts with a sample that is
within one fifth of one wavelength of the dielectric boundary.
Attenuated total reflection (ATR) spectroscopy is predicated on the
concept that, when light traveling within a medium impinges upon an
interface between that medium and a medium of lower refractive
index, it either passes into the second medium or is totally
internally reflected, depending on whether the quantity
[n.sub.1/n.sub.2 sin .theta..sub.i] is less than or greater than
one. In this relationship, n.sub.1 and n.sub.2 are the refractive
indices of the first and second media, respectively, and
.theta..sub.i is the angle of incidence. If n.sub.1/n.sub.2 sin
.theta..sub.1 is greater than one, total internal reflection
occurs. Although the internal reflection is referred to as total,
the light, during the reflection process, penetrates a short
distance into the second medium. The depth of penetration depends
in a predictable fashion on the refractive indices of the two media
and the angle of incidence, and is typically on the order of tenths
of the wavelength of the light. If the incident light includes a
wavelength absorbed by a constituent of the second medium, light of
such wavelength will be partially absorbed or attenuated during
reflection due to the penetration of the light into the second
medium. This effect is referred to as attenuated total reflection.
Due to the very shallow penetration of the light into the second
medium, ATR is a useful technique for measuring absorbance by
strongly absorbing materials. ATR has also been particularly useful
for measuring absorbance of material deposited on a surface.
Attenuated total reflection spectroscopy is widely used to collect
an absorption spectrum from samples that are too opaque for direct
absorption measurements.
In practice, one surface of an internal reflecting element (IRE) is
placed in contact with a test sample. An incident beam of radiation
is directed through the IRE so that it is totally internally
reflected at the boundary between the IRE and the test sample. Some
of the energy of the incident radiation is absorbed by the test
sample through evanescent coupling. The amount of absorption is
representative of the molecular structure and/or the molecular
species found in the test sample. The reflected radiation,
therefore, includes information from which an absorption spectrum
for the test sample can be acquired. IREs utilizing total internal
reflection or attenuated total reflection principles are commonly
found in optical systems designed to analyze samples by assessing
the optical constants of the sample and by establishing the
physical and chemical composition thereof. Examples of IREs
disposed in various optical systems are shown, for example, in U.S.
Pat. Nos. 4,602,869 and 3,393,603. In some embodiments, the IRE is
a germanium crystal or a zinc selenide crystal. The angle of
incidence is defined as the angle between the ray direction and the
normal to the surface. A 45-degree angle of incidence is often
convenient for a multi-pass FTIR-ATR element. However, the angle of
incidence and the composition of an element can be varied to
optimize the parameters for a given experiment.
In ATR-FTIR spectroscopy, light is totally internally reflected
inside a prism of high refractive index (FIG. 8, inset a). Photons
come out of the crystal penetrating the sample, and then are
coupled back into the system. This evanescent wave can interact
with the material on the surface of the crystal (diamond in this
case). The intensities of the frequencies of light measured after
passing through the prism are highly sensitive to the materials
present on the surface of the crystal. The penetration depth of the
photons, a function of the wavelength of light and the refractive
indices of the ATR crystal and sample, is about two microns at 1000
cm.sup.-1 wavenumber. The Bruker vertex 70 spectrometer can cover
the range from 15800 to 10 cm.sup.-1 with a spectral resolution
between 0.25 to 256 cm.sup.-1 and specific number of scan averages
can be selected as needed depending on the signal/noise ratio. The
serum sample can be deposited on the crystal surface and allowed to
air dry (approximately seven minutes) before obtaining the ATR
absorbance spectrum (FIG. 8). The specific features of the spectra
can be simulated by using Lorentzian oscillators corresponding to
the expected individual components. The amide I peak can be fitted
with oscillators 1, 2 and 3. Spectra can be normalized to the Amide
I peak (1642 cm.sup.-1) which is the commonly used standard in
spectroscopy studies involving biological samples. The amide I and
the polysaccharides regions can be simulated by using Lorentzian
oscillators corresponding to the expected individual components
(1,2,3 and 4,5,6,7) with the RMS error as low as 0.004. This can
allow one to match the colitic (polysaccharides) spectra with the
known concentrations.
Mathematical and statistical operations that are performed in the
course of practicing the present methods can be performed using any
suitable computational equipment and software. For example, a
commercially available personal computer can be used as a platform
for software that can facilitate the acquisition of data, the
calculation of difference spectra and perform spectral and other
analysis. Computers networked with an FTIR instrument can be
employed to acquire data on one machine and process it on another.
Suitable data acquisition and management software packages can be
designed and written de novo or can be purchased. Suitable
commercially available software packages can include SCANTRAQ
BASIC.TM. software package available from FTG Software Associates
of Princeton, N.J., and GRAMS/32.TM. Version 5.2 software package,
available from ThermoGalactic of Salem, N.H.
In some embodiments, the process of acquiring a spectrum of a
sample is automated.
Suitable commercially available software packages for automated
spectrum acquisition include the WINFIRST.TM. package available
from Thermo Mattson of Madison, Wis., and the AUTOPRO.TM. software
package available from Pike Technologies, Inc. of Madison, Wis.
These software packages can be employed to automate spectrum
acquisition and can be useful for analyzing large numbers of
samples. In some embodiments, the process is fully automated and
can comprise an autosampler to inject and remove samples and a
spectrum acquisition software package to run an FTIR microscope or
FTIR bench accessory. Additionally, the identified software
packages can be modified, or software can be written or purchased,
to perform the various mathematical and statistical operations that
can be performed when acquiring data by employing the present
inventive methods. For example, software can be provided and
employed to analyze an acquired spectrum, whereby the water
component is automatically subtracted from the spectrum and the
quality and quantity of secondary structure is subsequently
identified using algorithms referred to, incorporated and disclosed
herein. In this embodiment, a researcher can simply prepare the
autosampler, configure the software and begin the process.
Inflammatory abnormalities are a large group of disorders that
underlie a vast variety of human diseases. The immune system is
often involved with inflammatory disorders, demonstrated in both
allergic reactions and some myopathies, with many immune system
disorders resulting in abnormal inflammation. Non-immune diseases
with etiological origins in inflammatory processes include cancer,
atherosclerosis, and ischaemic heart disease.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory
disorder that primarily affects joints. It may result in deformed
and painful joints, which can lead to loss of function. The disease
may also have signs and symptoms in organs other than joints.
Inflammatory bowel disease (IBD) is a group of inflammatory
conditions of the colon and small intestine. Crohn's disease and
ulcerative colitis are the principal types of inflammatory bowel
disease. It is important to note that not only does Crohn's disease
affect the small intestine and large intestine, it can also affect
the mouth, oesophagus, stomach and the anus whereas ulcerative
colitis primarily affects the colon and the rectum.
In spite of Crohn's and UC being very different diseases, both may
present with any of the following symptoms: abdominal pain,
vomiting, diarrhea, rectal bleeding, severe internal cramps/muscle
spasms in the region of the pelvis and weight loss. Anemia is the
most prevalent extraintestinal complication of inflammatory bowel
disease. Associated complaints or diseases include arthritis,
pyoderma gangrenosum, primary sclerosing cholangitis, and
non-thyroidal illness syndrome (NTIS). Associations with deep vein
thrombosis (DVT) and Bronchiolitis obliterans organizing pneumonia
(BOOP) have also been reported.
Once the disclosed method indicates the presence of an IBD,
diagnosis can be confirmed by biopsy on colonoscopy.
Medical treatment of IBD is individualized to each patient. The
choice of which drugs to use and by which route to administer them
(oral, rectal, injection, infusion) depends on factors including
the type, distribution, and severity of the patient's disease, as
well as other historical and biochemical prognostic factors, and
patient preferences. For example, mesalazine is more useful in
ulcerative colitis than in Crohn's disease. Generally, depending on
the level of severity, IBD may require immunosuppression to control
the symptom, such as prednisone, TNF inhibition, azathioprine
(Imuran), methotrexate, or 6-mercaptopurine.
Often, anti-inflammatory steroids are used to control disease
flares and were once acceptable as a maintenance drug. In use for
several years in Crohn's disease patients and recently in patients
with ulcerative colitis, biologicals have been used such as TNF
inhibitors. Severe cases may require surgery, such as bowel
resection, strictureplasty or a temporary or permanent colostomy or
ileostomy. Ulcerative colitis can in most cases be cured by
proctocolectomy, however this may not eliminate extra-intestinal
symptoms. A small percentage of patients with ileo-anal pouches do
have to manage occasional or even chronic pouchitis. In Crohn's
disease, surgery involves removing the worst inflamed segments of
the intestine and connecting the healthy regions, but
unfortunately, it does not cure Crohn's or eliminate the disease,
as at some point after the first surgery, Crohn's disease recurs in
the healthy parts of the intestine, usually at the resection site.
(For example, if a patient with Crohn's disease has an ileocecal
anastomosis, in which the caecum and terminal ileum are removed and
the ileum is joined to the ascending colon, their Crohn's will
nearly always flare-up near the anastomosis or in the rest of the
ascending colon).
A relatively new treatment option is fecal bacteriotherapy (FBT),
which has been used to successfully treat IBD in several small
studies.
The term "subject" refers to any individual who is the target of
administration or treatment. The subject can be a vertebrate, for
example, a mammal. Thus, the subject can be a human or veterinary
patient. The term "patient" refers to a subject under the treatment
of a clinician, e.g., physician.
The term "sample from a subject" refers to a tissue (e.g., tissue
biopsy), organ, cell (including a cell maintained in culture), cell
lysate (or lysate fraction), or body fluid from a subject.
Non-limiting examples of body fluids include blood, urine, plasma,
serum, tears, lymph, bile, cerebrospinal fluid, interstitial fluid,
aqueous or vitreous humor, colostrum, sputum, amniotic fluid,
saliva, anal and vaginal secretions, perspiration, semen,
transudate, exudate, and synovial fluid.
A number of embodiments of the invention have been described.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of the
invention. Accordingly, other embodiments are within the scope of
the following claims.
EXAMPLES
Example 1
Minimally Invasive Screening for Colitis Using Attenuated Total
Internal Reflectance Fourier Transform Infrared Sectroscopy
Materials and Methods
Mice
Three week-old female C57BL/6 wild type (WT) and interleukin 10
knockout (IL10-/-) mice were obtained from Jackson Laboratories
(Bar Harbor, Me.). Toll-like receptor knockout (TLR5-/-) mice were
grown in our facility. Mice were group housed under a controlled
temperature (25.degree. C.) and photoperiod (12:12-h light-dark
cycle) and fed ad libitum. All studies were performed in accordance
with the Institutional Animal Care and Use Committee at Georgia
State University (Atlanta, Ga.), permit number: A14010.
Development of Colitis in IL10-/-
IL10-/- mice develop colitis on a time dependent manner In order to
assess the intestinal inflammation in those mice at different times
of colitis development, feces were collected at week 4 and week 14
to measure Lcn-2. Blood was collected at week 4 and 14 to obtain
sera by centrifugation using serum separator tubes (BD Biosciences,
Franklin Lakes, N.J.)
Dextran Sodium Sulphate (DSS) Induced Colitis
C57BL/6 WT mice were administered DSS (MP Biomedicals, Solon, Ohio)
at 3% in drinking water ad libitum for 7 days. Feces and blood were
collected at day 0 (before DSS treatment) and day 7. Hemolysis-free
serum was collected by centrifugation using serum separator tubes.
Mice were sacrificed by CO.sub.2 euthanasia.
Collagen Antibody-Induced Arthritis Model
BALB/C WT mice received collagen antibodies injections (200 .mu.L)
on day 0 by an intravenous injection (tail vein). On day 6, mice
received a lipopolysaccharide (LPS) boost injection (200 .mu.L) by
intraperitoneal injection. Blood samples were collected from each
mouse on pretreatment (day -2) and on day 12 from the jugular vein.
Hemolysis-free serum was collected by centrifugation using serum
separator tubes.
TLR5-/- Model of Metabolic Syndrome
TLR5-/- spontaneously develop metabolic syndrome as previously
described (Vijay-Kumar, M., et al., Science, 2010.
328(5975):228-231). Age-matched WT and TLR5-/- mice were fasted for
5-h and baseline blood glucose levels measured with a blood glucose
meter (Roche) using blood collected from the tail vein.
H&E Staining of Colonic Tissue
Mouse colons were fixed in 10% buffered formalin for 24 hours at
room temperature and then embedded in paraffin. Tissues were
sectioned at 5-.mu.m thickness and stained with hematoxylin &
eosin (H&E) using standard protocols. Images were acquired
using a Zeiss Axioskop 2 plus microscope (Carl Zeiss Microlmaging)
equipped with an AxioCam MRc5 CCD camera (Carl Zeiss).
Quantification of Fecal and Serum Lcn-2 by ELISA
Fecal samples were reconstituted in PBS containing 0.1% Tween 20
(100 mg/me. After centrifugation, clear supernatants were
collected. Serum samples were diluted in kit-recommended reagent
diluent (1.0% BSA in PBS). Lipocalin-2 (Lcn-2) levels were
estimated in the supernatants and/or serum using Duoset murine
Lcn-2 ELISA kits (R & D Systems, Minneapolis, Minn.).
Colonic Myeloperoxidase (MPO) Assay
Neutrophil influx in colon was analyzed as marker of inflammation
by assaying the enzymatic activity of MPO, a neutrophils marker.
Briefly, tissue (50 mg/mL) was thoroughly washed in PBS and
homogenized in 0.5% hexadecyltrimethylammonium bromide (Sigma, St.
Louis, Mo.) in 50 mM PBS, (pH 6.0), freeze-thawed 3 times,
sonicated and centrifuged. MPO was assayed in the clear supernatant
by adding 1 mg/mL of dianisidine dihydrochloride (Sigma) and
0.0005% H.sub.2O.sub.2 and the change in optical density measured
at 450 nm. Human neutrophil MPO (Sigma) was used as standard. One
unit of MPO activity was defined as the amount that degraded 1 mmol
peroxidase per minute.
Fourier Transform Infrared (FTIR) Spectroscopy
A Bruker Vertex 70 FTIR spectrometer was used to obtain all the
spectroscopic results. The samples were scanned covering the
wavelength range of 4000 to 400 cm.sup.-1 and the 1800 to 1000
cm.sup.-1 section was used for this study. A medium Blackman-Harris
appodization was employed with a resolution of 8 cm.sup.-1. The
samples were scanned 50 times and averaged. Each co-added sample
scan was repeated 5 times and averaged. A room temperature
Deuterated Lanthanum Alanine doped TriGlycine Sulphate (DLaTGS)
pyroelectric detector was employed. The infrared light beam
intensity was controlled by passing it through a 3 mm aperture.
This is done to optimize the detector response and prevent
saturation. A Parker-Balston dry air purging system was used to
reduce the moisture and carbon dioxide levels of the ambient air in
the spectrometer.
Attenuated Total Reflectance (ATR) Configuration
MVP-Pro ATR accessory from Harrick-Scientific was used for all
spectroscopic measurements in this study. A diamond crystal (1
mm.times.1.5 mm) was the internal reflection element configured to
have a single reflection of the infrared radiation. A sample of one
microliter is deposited on the crystal surface and allowed to air
dry (.about.5 minutes). An evanescent wave with an approximate
penetration depth of 2 microns (dependent on the refractive indices
of the ATR crystal and sample and the wavelength of light)
interacts with the sample. The output spectra is an ATR absorbance
spectra which is subsequently analysed.
Post Processing Techniques
The 5 reads of the 50 co-added scans for each sample (total of 250
scans) are averaged. The spectra were sectioned to the 1800 to 1000
cm.sup.-1 range. Using OPUS 7.2 software, all the spectra were
internally normalized (Yu, C. and J. Irudayaraj, Biopolymers, 2005.
77(6):368-377) by scaling the entire sectioned range so that the
absorbance value at the 1642 cm.sup.-1 peak (Amide I) was 2.0.
Spectral deconvolution was also done to better resolve the peaks by
obtaining the second derivative followed by a 9 point smoothing
using Microsoft Excel software.
Data Analysis Techniques
Cluster and heterogeneity analyses were carried out in the spectral
range of 1140 to 1000 cm.sup.-1 using the Bruker Optics OPUS 7.2
software. The algorithm calculates the Euclidean distance between
each spectrum and groups them into clusters based on the conformity
of the spectra with each other. The resulting data is plotted as a
heterogeneity dendrogram chart where the heterogeneity index on the
y-axis indicates the degree of heterogeneity between the identified
clusters. Student's t-tests were carried out for the DSS study and
not for the IL10-/-, CAIA and Metabolic syndrome studies due to the
smaller sample sizes, although the uncertainty levels of the
averages are shown.
Results
DSS-induced colitis (Laroui, H., et al., PloS one, 2012.
7(3):e32084; Chassaing, B., et al., Current Protocols in
Immunology:15.25.1-15.25.14) is a commonly used chemically-induced
mouse model of acute colitis which has similarities to ulcerative
colitis in human DSS first disrupts the intestinal barrier
functions followed by an increase of inflammation which closely
resembles histological and clinical characteristics of IBDs such as
ulcerative colitis (Clapper, M. L., et al., Acta pharmacologica
Sinica, 2007. 28(9):1450-1459; Pe e, M. and A. Cerar, Journal of
biomedicine & biotechnology, 2011. 2012:718617-718617). The
second model studied, IL10-/- mouse model (Kennedy, R., et al.,
British journal of surgery, 2000. 87(10):1346-1351) closely
resembles the physiological, histological and biochemical features
of human chronic colitis and develops colitis mediated by T helper
cell 1 (Th1) cells. Mice with targeted deletion of the IL10 gene
spontaneously develop chronic enterocolitis with massive
infiltration of lymphocytes, activated macrophages, and neutrophils
in a Th1 cell-mediated mechanism (Kim, J. J., et al., Journal of
Visualized Experiments: JoVE, 2012(60):3678). The predictability of
the timing of colitis in IL10-/- mice allows longitudinal
assessment of blood samples during colitis progression from 4 weeks
(no symptoms shown) up to 14 weeks, the age at which the mice
display signs of severe colitis.
To confirm the effectiveness of these two models as tools for
investigating spectral markers for colitis, the development of
colitis was assessed in these mice using other established
techniques. Histological features were assessed by H&E
staining, and the degree of inflammation was measured in DSS and
IL10-/- model by respectively assessing MPO activity, a marker of
inflammation in the colon (Viennois, E., et al., Laboratory
Investigation, 2014. 94(9):950-965), and measuring fecal Lipocalin
2 (Lcn-2) levels, previously described (Chassaing, B., et al., PloS
one, 2012. 7(9):e44328) as being a robust fecal marker that
correlates with the severity of inflammation. MPO is produced by
neutrophils, a class of leukocytes that highly infiltrate into the
mucosa in a situation of intestinal inflammation. Increases of
Lcn-2 levels and MPO activity in the feces of IL10-/- mice (FIG.
1A) and in DSS-induced colitis colon samples (FIG. 1B)
respectively, were observed. The increase of lymphocyte
infiltration (FIG. 1C, arrow head) and the erosion (FIG. 1c, arrow)
of intestinal glands (crypt), observed on the H&E stained
picture of the colon confirmed that, in contrast to the control
groups (non-colitic), the DSS-treated and the IL10-/- mice develop
colitis.
Spectroscopic measurements were performed on sera from DSS-induced
colitis mice compared to the same mice before intake of DSS
(control mice) and on colitic IL10-/- mice compared to the same
mice before the development of colitis. Serum was chosen due to its
stability and absence of any additives such as anticoagulants.
Serum samples were deposited on the ATR crystal and allowed to dry.
By allowing the water in the sera to evaporate, the signal to noise
ratio of the spectral signal of other sera components are greatly
enhanced, which are otherwise occluded by the broad water
absorption. Similar significant differences in absorbance were
observed in both DSS (FIG. 2) and IL10-/- (FIG. 3) mouse models
between the control groups (non-colitic) and the colitic groups at
.about.1033 cm.sup.-1 and .about.1076 cm.sup.-1. Both absorbance
peaks have been attributed to the symmetric stretching modes of
C--O indicating the presence of saccharides (Movasaghi, Z., et al.
Applied Spectroscopy Reviews, 2008. 43(2):134-179), with the
vibrational modes at .about.1033 cm.sup.-1 and .about.1076
cm.sup.-1 due to glucose and mannose respectively (Petibois, C., et
al., Clinical chemistry, 1999. 45(9):1530-1535).
It has been reported that in colitis serum samples, there is a
reduction in butyrate oxidation with a compensatory increase in the
oxidation levels of glucose (Ahmad, M., et al., Gut, 2000.
46(4):493-499). Hence, the increase in the absorbance at
.about.1033 cm.sup.-1 in colitic serum samples could be an
indication of colitis.
Studies in humans have shown the co-occurrence of ulcerative
colitis with that of diabetes and glucose intolerance (Maconi, G.,
et al., World Journal of Gastroenterology: WJG, 2014.
20(13):3507-3515). In order to exclude the possibility that the
mannose and glucose peaks obtained for the IL10-/- and DSS-induced
models of colitis originate from the co-occurrence of other glucose
intolerance conditions, similar assays were performed using a mouse
model developing metabolic syndrome. Mice deficient of Toll-like
receptor 5, a component of the innate immune system that is
expressed in the intestinal mucosa, exhibit hyperphagia and develop
the hallmark features of metabolic syndrome, including
hyperlipidemia, hypertension, insulin resistance, and increased
adiposity (Vijay-Kumar, M., et al., Science, 2010.
328(5975):228-31).
As seen in FIG. 4, metabolic syndrome samples did not show any
significant differences in absorbance at the .about.1033 cm.sup.-1
and .about.1076 cm.sup.-1 peaks with respect to ATR-FTIR
spectroscopy in this wavelength range of interest. This indicates
that these particular mannose and glucose peaks observed in colitic
samples were not a result of metabolic syndrome.
The next objective was to determine whether the absorbance changes
in the two peaks at .about.1033 cm.sup.-1 and .about.1076
cm.sup.-1, were specific to intestinal inflammation or associated
with any kind of inflammation. Collagen antibody-induced arthritis
(CAIA) was employed as a model of extra-intestinal inflammation. An
increase in absorbance was seen in arthritic sera samples a
.about.1033 cm.sup.-1 (similar to colitic samples), but not at
.about.1076 cm.sup.-1 as previously seen in the inset of FIG. 4.
This result suggests that the glucose peak might not be specific to
colitis but general to an inflammation from any origin. However,
the mannose peak at .about.1076 cm.sup.-1 appeared to be specific
to colitis. It has been reported that in ulcerative colitis cases
in humans, one of the glycoprotein fractions in the colonic mucus
has elevated levels of mannose that was confirmed using biological
assays (Teague, R., et al. BMJ, 1973. 2(5867):645-646). The lesions
on the colon characteristic of colitis can facilitate the diffusion
of mannose into the circulating blood stream, thus manifesting as
increased levels of mannose in serum. This phenomenon could explain
the increased levels of mannose in the colitic mice serum samples
in the DSS model at .about.1076 cm.sup.-1 spectral marker. Another
study using Proton Nuclear Magnetic Resonance spectroscopy reports
that there is a significant increase in mannose levels (Schicho,
R., et al., Journal of Proteome Research, 2010. 9(12):6265-6273) in
the serum for DSS-induced colitic mice which is confirmed by our
ATR-FTIR spectroscopic study.
As seen in FIG. 5A, the absorbance levels at .about.1033 cm.sup.-1
indicated that the glucose peak increased at the onset of arthritis
and colitis. The absorbance data points for the metabolic samples
did not show a clear separation from the normal in either
individual (FIGS. 5A and B) or the average (FIGS. 5C and D) values
at .about.1033 and .about.1076 cm.sup.-1. The error bars associated
with the averaged absorbance values of diseased samples in FIGS. 5c
and d were larger than the normal sample values as each mouse could
be at a different stage of the disease.
The absorbance data for arthritis also showed a separation at
.about.1033 cm.sup.-1 but no appreciable difference in the mannose
peak at .about.1076 cm.sup.-1 (FIG. 5B). However, especially for
colitis samples, there were clear separations from the normal
samples.
Moreover, arthritis serum samples displayed an absorption peak at
1292 cm.sup.-1 which was observed only for arthritis and not for
colitis (both DSS and IL10-/-) or metabolic syndrome serum samples.
This peak was identified as thymine (Movasaghi, Z., et al. Applied
Spectroscopy Reviews, 2008. 43(2):134-179). It has been reported
that, in cases of arthritis, thymidine begins to break down to
thymine (Nykanen, P., Scandinavian journal of immunology, 1979.
9(5):477-482) which explains the increased presence of thymine in
the serum.
On deconvolution of the spectra by performing the second derivative
on the absorbance values (FIG. 6), one can clearly distinguish
between the serum samples representative of intra-(colitic) and
extra-(arthritic) intestinal inflammation based on the thymine
peak.
There was no notable difference in absorbance values for the
metabolic syndrome samples and their controls indicating that the
presence of metabolic syndrome was not manifested at these spectral
markers. The analysis indicated that the increase in glucose peak
(1033 cm.sup.-1) was common to colitis and arthritis, but the
increase in mannose peak (1076 cm.sup.-1) was unique to
colitis.
Cluster and heterogeneity analyses, commonly employed in
computational biology, were carried out in the spectral range of
1140 to 1000 cm.sup.-1 to include the glucose (1033 cm.sup.-1) and
mannose (1076 cm.sup.-1) peaks. The input datasets include the 12
DSS induced colitic and 12 control sample spectra. The resulting
data is plotted as a heterogeneity dendrogram chart (FIGS. 7 and 8)
indicating that the spectra were correctly grouped together and
classified into two clusters, namely control and colitic with a
high degree of heterogeniety.
Conclusion
A rapid, simple, cost effective and minimally invasive technique,
ATR-FTIR spectroscopy, has been demonstrated as an effective tool
to detect colitis in mice serum. The use of a metabolic syndrome
mouse model and an arthritis model indicate the specificity of the
mannose peak for colitis. A portable device capable of detecting
similar variations in mannose and glucose absorbance will require a
specific infrared detector capable of simultaneous multiband
detection in order to avoid bulky interferometers or gratings. The
developments in infrared detector technology allowing room
temperature operation of multiband infrared detectors make this
possible (Perera, A. G. U., et al., Microelectronics Journal, 2009.
40(3):507-511; Jayaweera, P. V. V., et al. Applied Physics Letters,
2007. 91(6):063114; Ariyawansa, G., et al., Infrared Physics &
Technology, 2007. 50(2-3):156-161; Perera, A., et al., Applied
Physics Letters, 2006. 89(13):131118). This technology can be
further developed into a personalized diagnostic tool in which
patient-to-patient differences in molecular signatures would allow
the assessment of disease status and personalized drug management.
This technology could be integrated in a portable device, like the
current glucometer, that each patient would wear as a platform to
monitor multiple health parameters at the point-of-care,
facilitating the creation of bedside technologies for diagnostics
and treatment monitoring for various other medical conditions
(Titus, J., et al., Applied Physics Letters, 2014. 104(24):243705)
such as arthritis, viral or bacterial infections, allergies etc
including IBD.
Example 2
Screening for Colitis Using (Infrared) Spectroscopic Signatures
Spectroscopic measurements were performed on sera from DSS-induced
colitis mice compared to the same mice before intake of DSS
(control mice). Spectral deconvolution was performed on the amide I
region by taking the second derivative of the absorbance. As seen
on FIG. 9, two major peaks (dips in second derivative) are observed
at 1635 and 1650 cm.sup.-1 which are assigned as beta sheet and
alpha helix (Movasaghi, Z., et al., Applied Spectroscopy Reviews
2008, 43:134-179) respectively which are components of proteins.
The alpha helix to beta sheet ratio is always higher in normal
serum samples compared to the DSS induced colitis samples. This can
be connected to the selective upregulation/downregulation of
certain proteins that are determined as markers (Viennois, E. et
al., Journal of proteomics 2015, 112:166-179) for colitis.
Arthritis being an extra-intestinal inflammatory model serves as a
control for colitis. There is no significant difference in the
aforementioned ratios indicating its uniqueness to colitis.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
* * * * *